Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. Uba1 was thought to be the only E1 until the recent identification of Uba6. To differentiate the biological functions of Uba1 and Uba6, we applied an orthogonal ubiquitin transfer (OUT) technology to profile their ubiquitination targets in mammalian cells. By expressing pairs of an engineered ubiquitin and engineered Uba1 or Uba6 that were generated for exclusive interactions, we identified 697 potential Uba6 targets and 527 potential Uba1 targets with 258 overlaps. Bioinformatics analysis reveals substantial differences in pathways involving Uba1- and Uba6-specific targets. We demonstrate that polyubiquitination and proteasomal degradation of ezrin and CUGBP1 require Uba6, but not Uba1, and that Uba6 is involved in the control of ezrin localization and epithelial morphogenesis. These data suggest that distinctive substrate pools exist for Uba1 and Uba6 that reflect non-redundant biological roles for Uba6.